Cell-permeable ceramides

This is in agreement with data showing that natural ceramides do not have any effect on CT activity in vitro (Sohal and Cornell, 1990). The mechanism by which cell-permeable ceramides exert their effect on CPT and EFT activity and whether natural or intracellular generated ceramides have a similar effect on phospholpid synthesis is at present unknown. 

Treatment of Rat-2 fibroblasts with various cell-permeable ceramides resulted, as discussed above, in a rapid inhibition of phospholipid synthesis. An intriguing observation was that C2-ceramides inhibited PC biosynthesis without affecting the synthesis of PE via the CDP-ethanolamine route. C2-ceramide are well known inducers of apoptosis and inhibit DNA- and protein synthesis (Obeid et al, 1993 Jayadev et al, 1995). C2-ceramide induced apoptosis in the rat-2 fibroblasts (Houweling et al, unpublished data), suggesting that inhibition of PE biosynthesis is not a prerequisite for apoptosis. 

For example, Jurkat T cells apoptose in response to serum withdrawal, addition of exogenous cell-permeable ceramide or stimulation with the apoptotic agonist Fas. However, transfection with SPHKl, measured as a 400-600-fold increase in activity, reduced apoptosis and inaeased proliferation (Ohvera et al, 1999). These effects were correlated with an inhibition of the activity of caspase 3, an initiator caspase (Olivera et al, 1999), A similar effect has recently been observed in rat pheochromocytoma PC12 cells (Edsall etal, 2(X)1). Tropbic withdrawal led to the activation of... 

Recently, it has been shown that cell-permeable ceramides dramatically inhibited the synthesis of the two major membrane phospholipids, PC and PE (Bladergroen et al, 1999b Allan, 2000). The inhibition of phospholipid synthesis was rapid, within 2 h, and resulted in massive apoptosis after 16-24 h. The mechanism by which short-chain ceramides exert their effect on phospholipid synthesis is possibly cell type dependent. In baby-hamster kidney (BHK) fibroblasts PC synthesis was reduced at the level of CT, the putative rate-determining enzyme in the CDP-choline pathway (Allan, 2000). This conclusion was based solely on radio-label studies in combination with an earlier published observation (Wieder et al., 1995) showing that C2-SM (the SM generated from C2-ceramide by SM synthase, which was actively synthesized in the BHK-cells) inhibited CT activity in vitro. On the other hand, data obtained from studies with rat-2 fibroblasts clearly showed that short-chain ceramides regulate the synthesis of PC and PE mainly at the final step of the CDP-pathways. This conclusion was based on the following observations (a) incorporation of [3H]-choline into PC and... 

One of the first reports showing that DAG attenuates ceramide induced apoptosis used two human myeloid leukemia cell lines, namely HL-60 and U937. Exposure of HL-60 and U937 cells to SMase or lOpM cell-permeable ceramides for 6 h resulted in DNA-laddering, a typical hallmark of apoptosis. This could be abolished by di-octanoylglycerol, a cell permeable... 

Houwehng, M., 1999b, Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides. Eur. J. Biochem. 264 152-160... 

Abousalham A, Hobman TC, Dewald J, Garbutt M, Brindley DN. Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin- Darby canine kidney cells by preventing the membrane association of ADP- ribosylation factor. Biochem J 361(2002) 653-661. 

Ferrying of molecules into cells via entry through caveolae may represent a way to traffic specifically cytotoxic molecules to specific action sites. For example, elevating the intracellular level of the sphingolipid ceramide is known to exert antimitogenic and proapoptotic effects. While ceramide is cell-permeable and displays antiapoptotic properties in vitro, systemic in vivo use of ceramide is hampered by its hydrophobicity. Using a C6-ceramide formulation in pegylated liposomes was shown to elicit a sixfold reduction in solid phase tumors, when compared to unloaded liposomes in a mouse model of breast adenocarcinoma [68],... 

Conformational restriction of ceramide led to the development of cell-permeable, indirectly acting inhibitors of glycosyltransferases. Together with other ceramide analogs [10, 36] this concept demonstrates that, in principle, it is pharmacologi-... 

Other laboratories have also shown that enzymes of the CDP-choline pathway for the production of PC in the cells, including CPT, show reduced activity when cells are incubated with cell-permeable C2/C6 ceramides (Bladergroen et al., 1999 Ramos et al., 2002), and it has been predicted that this inhibition may be due to the competitive inhibition by ceramides owing to the similarity in the stmcture to one of the substrates for CPT, DAG. 

Once apoptosis is triggered, a stereotyped sequence of premitochondrial events occurs that executes the cell death process. In many cases proteins and/or lipid mediators that induce changes in mitochondrial membrane permeability and calcium regulation are produced or activated. For example, the pro-apoptotic Bcl-2 family members Bax, Bad and Bid may associate with the mitochondrial membrane and modify its permeability. Membrane-derived lipid mediators such as ceramide and 4-hydroxynonenal can also induce mitochondrial membrane alterations that are critical for the execution of apoptosis. 

The third class of lipids found in stratum corneum extracts is represented by cholesterol and cholesteryl esters. The actual role of cholesterol remains enigmatic, and no clear reason for its role in the barrier function has been proposed so far. However, it is possible that contrary to what is the role in cell membranes where cholesterol increases close packing of phospholipids, it can act as kind of a detergent in lipid bilayers of long-chain, saturated lipids.30,31 This would allow some fraction of the barrier to be in a liquid crystalline state, hence water permeable in spite of the fact that not only ceramides, but also fatty acids found in the barrier are saturated, long-chain species.28,32... 

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